1. Field of the Invention
The present invention relates to a sensor, utilizing an evanescent wave, which analyzes a sample by causing a light beam to reflect at the interface between a thin film layer in contact with the sample and a dielectric block to generate an evanescent wave and then measuring a change in the intensity of the totally reflected light beam due to the evanescent wave.
2. Description of the Related Art
A surface plasmon resonance sensor is known as one utilizing an evanescent wave. If free electrons vibrate collectively in a metal, a compression wave called a plasma wave will be generated. The compression wave, generated in the metal surface and quantized, is called a surface plasmon. The surface plasmon resonance sensor is used to analyze the properties of a sample by taking advantage of a phenomenon that the surface plasmon is excited by a light wave. Various types of sensors have been proposed. Among such sensors, one employing the “Kretschmann configuration” is particularly well known (e.g., see Japanese Unexamined Patent Publication No. 6 (1994)-167443).
The surface plasmon resonance sensor employing the aforementioned “Kretschmann configuration” is constructed basically of (1) a dielectric block formed into the shape of a prism; (2) a metal film, formed on one surface of the dielectric block, for placing a sample thereon; (3) a light source for emitting a light beam; (4) an optical system for making the light beam enter the dielectric block at various angles of incidence so that a condition for total internal reflection is satisfied at the interface between the dielectric block and the metal film; (5) photodetection means for detecting the intensity of the light beam totally reflected at the interface; and (6) measurement means for measuring the state of surface plasmon resonance (SPR) on the basis of the result of detection obtained by the photodetection means.
In order to obtain various angles of incidence in the aforementioned manner, a relatively thin light beam may be caused to strike the above-described interface while changing the angle of incidence, or a relatively thick convergent or divergent light beam may be caused to strike the interface so that it has incident components at various angles. In the former, a light beam whose angle of reflection varies with a change in the angle of incidence can be detected by a small photodetector movable in synchronization with a change in the angle of reflection, or by an area sensor extending in the direction where the angle of reflection varies. In the latter, on the other hand, light beams reflected at various angles can be detected by an area sensor extending in a direction where the reflected light beams can be all received.
In the above-described surface plasmon resonance sensor, if a light beam strikes a metal film at a specific incidence angle θsp greater than a critical incidence angle at which total internal reflection (TIR) takes place, an evanescent wave having electric field distribution is generated in a sample in contact with the metal film. With the evanescent wave, the above-described surface plasmon is excited in the interface between the thin film layer and the liquid sample. When the wave number vector of the evanescent wave is equal to the wave number of the surface plasmon and therefore the wave numbers between the two are matched, the evanescent wave resonates with the surface plasmon and the light energy is transferred to the surface plasmon. As a result, the intensity of the light totally reflected at the interface between the dielectric block and the metal film drops sharply. This sharp intensity drop is generally detected as a dark line by the above-described photodetection means.
Note that the aforementioned resonance occurs only when the incident light beam is p-polarized light. Therefore, it is necessary to make settings in advance so that the incident light beam can strike the aforementioned interface as p-polarized light.
If the wave number of the surface plasmon is found from a specific incidence angle θsp at which attenuated total reflection (ATR) takes place (the angle θsp will hereinafter be referred to as an attenuated total reflection angle θsp) the dielectric constant of a sample to be analyzed can be calculated by the following Equation:
            K      sp        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              where Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in vacuum, and εm and εs represent the dielectric constants of the metal and the sample, respectively.
If the dielectric constant εs of the sample is found, the concentration of a specific substance in the sample is found based on a predetermined calibration curve, etc. As a result, the dielectric constant of the sample, that is, the properties of the sample related to the refractive index, can be found by finding the attenuated total reflection angle θsp.
In addition, a leaky mode sensor is known as a similar sensor making use of an evanescent wave (for example, see “Spectral Researches,” Vol. 47, No.1 (1998), pp. 21 to 23 and pp. 26 to 27). This leaky mode sensor consists basically of (1) a dielectric block formed into the shape of a prism; (2) a cladding layer formed on one surface of the dielectric block; (3) an optical waveguide layer, formed on the cladding layer, for placing a sample thereon; (4) a light source for emitting a light beam; (5) an optical system for making the light beam enter the dielectric block at various angles of incidence so that a condition for total internal reflection is satisfied at the interface between the dielectric block and the cladding layer; (6) photodetection means for measuring the intensity of the light beam totally reflected at the interface; and (7) measurement means for measuring the excited state of a waveguide mode on the basis of the result of detection obtained by the photodetection means.
In the above-described leaky mode sensor, if a light beam strikes the cladding layer through the dielectric block at an incidence angle greater than a critical incidence angle at which total internal reflection (TIR) takes place, the light beam is transmitted through the cladding layer. Thereafter, in the optical waveguide layer formed on the cladding layer, only light with a specific wave number, incident at a specific incidence angle, propagates in a waveguide mode. If the waveguide mode is excited in this manner, most of the incident light is confined within the optical waveguide layer, and consequently, ATR occurs in which the intensity of light totally reflected at the aforementioned interface drops sharply. The wave number of the light propagating through the optical waveguide layer depends upon the refractive index of the sample on the optical waveguide layer. Therefore, the refractive index of the sample and the properties of the sample related to the refractive index can be analyzed by finding the attenuated total reflection angle θsp at which ATR occurs.
In the field of pharmaceutical research, the above-described surface plasmon resonance sensor and leaky mode sensor are sometimes used in a random screening method for detecting a specific substance that is coupled to a sensing substance. In this case, the sensing substance is fixed on the aforementioned thin film layer (which is the aforementioned metal film in the case of surface plasmon resonance sensors, or the cladding layer and optical waveguide layer in the case of leaky mode sensors). Then, a liquid sample containing various target substances is supplied onto the sensing substance. And each time a predetermined time elapses, the attenuated total reflection angle θsp is measured. If a target substance in the liquid sample is a substance that is coupled to the sensing substance, then the coupling will cause the refractive index of the sensing substance to vary with the lapse of time. Therefore, every time a predetermined time elapses, the attenuated total reflection angle θsp is measured. Based on the measured value, it is measured whether or not a change has occurred in the attenuated total reflection angle θsp. Based on this result, it can be judged whether or not the target substance has been coupled to the sensing substance. That is, it can be judged whether or not the target substance is a specific substance that is coupled to the sensing substance. Examples of such a combination of a specific substance and a sensing substance are a combination of an antigen and an antibody, and a combination of an antibody and an antibody. A typical example of the measurement of such a combination is detection of the coupling between a human IgG (immunoglobulin G) antibody (which is a target substance) and a rabbit antihuman IgG antibody (which is a sensing substance) and a quantitative analysis thereof.
Note that in order to measure the coupled state between a target substance in a liquid sample and a sensing substance, the attenuated total reflection angle θsp itself does not always need to be detected. For example, a liquid sample with a target substance is added to a sensing substance. Then, a change in the attenuated total reflection angle θsp is measured. Based on the magnitude of the change, the coupled state between the target substance and the sensing substance can be measured.
In addition, the present inventors have proposed a sensor that is used to measure the above-described state of ATR by employing a measuring chip in the shape of a well which is easy to handle in making a measurement (e.g., Japanese Unexamined Patent Publication No. 2002-296172). In the case of a liquid sample, for example, a measurement can be made if only a small quantity of a liquid sample is supplied within the measuring chip. In addition, by employing a turntable capable of holding a plurality of measuring chips, a wide variety of samples can be measured in a short time.
In the sensor utilizing an evanescent wave, there are various methods of analyzing a sample by detecting the intensity of a light beam totally reflected at the aforementioned interface with photodetection means. For instance, a light beam is caused to strike the aforementioned interface at various angles of incidence so that a condition for total internal reflection (TIR) is satisfied at the interface. Then, the intensity of the light beam totally reflected at the interface is measured at each position corresponding to each incidence angle. Next, by detecting the position (attenuated total reflection angle θsp) of a dark line produced due to attenuated total reflection (ATR), the state of ATR is measured. In this way, the properties of a sample held by the measuring chip of the sensor may be analyzed. In addition, a light beam with a plurality of wavelengths is caused to enter a measuring chip at angles of incidence so that a total internal reflection condition is satisfied at the interface. Then, the intensity of the light beam totally reflected at the interface is measured for each wavelength. Next, by measuring the degree of ATR for each wavelength, the properties of a sample held by the measuring chip may be analyzed (see D. V. Noort, K. Johansen, C. -F. Mandenius, Porous Gold in Surface Plasmon Resonance Measurement, EUROSENSORS XIII, 1999, pp. 585–588).
Furthermore, a light beam is caused to enter a measuring chip at angles of incidence so that a total internal reflection condition is satisfied at the aforementioned interface. Then, the light beam is split into two light beams before the light beam strikes the interface. One of the two light beams is caused to interfere with the other light beam totally reflected at the interface. Next, by measuring the intensity of the light beam after the interference, the properties of a sample held by the measuring chip may be analyzed (see P. I. Nikitin, A. N. Grigorenko, A. A. Beloglazov, M. V. Valeiko, A. I. Savchuk, O. A. Savchuk, Surface Plasmon Resonance Interferometry for Micro-Array Biosensing, EUROSENSORS XIII, 1999, pp. 235–238).
As a sensor that analyzes the properties of a sample by utilizing an evanescent wave, there is known a sensor which makes a measurement by continuously supplying a liquid sample onto a sensing substance fixed on a measuring chip in the shape of a flat plate, using a flow passage mechanism. In measuring the coupled state between a sensing substance and a specific substance, a new liquid sample is always supplied onto the measuring chip. Therefore, since the concentration of a target substance in the liquid sample is kept constant, the coupled state can be accurately measured. In addition, in the case where the coupled state between the sensing substance and the specific substance is measured, and the coupling between the two has been performed, the decoupled state between the two can be measured by supplying a buffer solution (which is a liquid sample containing no specific substance) to the coupled body fixed to the measuring chip. Furthermore, in the case where gas is employed as a sample, or the case where a liquid sample containing gas is employed, the sample can be easily supplied onto the measuring chip by the flow passage mechanism.
Thus, various advantages are obtained by continuously supplying a liquid sample. However, a large quantity of the liquid sample must be prepared to continuously supply the liquid sample onto the measuring chip, or it is difficult to measure a wide variety of samples in a short time. Because of this, a user cannot perform a desirable measurement unless the user uses both a sensor which employs a measuring chip in the shape of a well, and a sensor equipped with a measuring chip in the shape of a flat plate and a flow passage mechanism.